Brown planthopper (BPH) is one of the most destructive insects affecting rice (Oryza sativa L.) production. Phenylalanine ammonialyase (PAL) is a key enzyme involved in plant defense against ...pathogens, but the role of PAL in insect resistance is still poorly understood. Here we show that expression of the majority of PALs in rice is significantly induced by BPH feeding. Knockdown of OsPALs significantly reduces BPH resistance, whereas overexpression of OsPAL8 in a susceptible rice cultivar significantly enhances its BPH resistance. We found that OsPALs mediate resistance to BPH by regulating the biosynthesis and accumulation of salicylic acid and lignin. Furthermore, we show that expression of OsPAL6 and OsPAL8 in response to BPH attack is directly up-regulated by OsMYB30, an R2R3 MYB transcription factor. Taken together, our results demonstrate that the phenylpropanoid pathway plays an important role in BPH resistance response, and provide valuable targets for genetic improvement of BPH resistance in rice.
Phenylketonuria (PKU) is caused by phenylalanine hydroxylase (PAH) deficiency that results in phenylalanine (Phe) accumulation. Pegvaliase, PEGylated recombinant Anabaena variabilis phenylalanine ...ammonia lyase (PAL), converts Phe to trans-cinnamic acid and ammonia, and is a potential enzyme substitution therapy to lower blood Phe in adults with PKU.
Two Phase 3 studies, PRISM-1 and PRISM-2, evaluated the efficacy and safety of pegvaliase treatment using an induction, titration, and maintenance dosing regimen in adults with PKU. In PRISM-1, pegvaliase-naïve participants with blood Phe >600 μmol/L were randomized 1:1 to a maintenance dose of 20 mg/day or 40 mg/day of pegvaliase. Participants in PRISM-1 continued pegvaliase treatment in PRISM-2, a 4-part clinical trial that includes an ongoing, open-label, long-term extension study of pegvaliase doses of 5 mg/day to 60 mg/day.
Of 261 participants who received pegvaliase treatment, 72.0% and 32.6% reached ≥12 months and ≥ 24 months of study treatment, respectively, and 65% are still actively receiving treatment. Mean (SD) blood Phe was 1232.7 (386.4) μmol/L at baseline, 564.5 (531.2) μmol/L at 12 months, and 311.4 (427) μmol/L at 24 months, a decrease from baseline of 51.1% and 68.7%, respectively. Within 24 months, 68.4% of participants achieved blood Phe ≤600 μmol/L, 60.7% of participants achieved blood Phe ≤360 μmol/L, below the upper limit recommended in the American College of Medical Genetics and Genomics PKU management guidelines, and 51.2% achieved blood Phe ≤120 μmol/L, below the upper limit of normal in the unaffected population. Improvements in neuropsychiatric outcomes were associated with reductions in blood Phe and were sustained with long-term pegvaliase treatment. Adverse events (AEs) were more frequent in the first 6 months of exposure (early treatment phase) than after 6 months of exposure (late treatment phase); 99% of AEs were mild or moderate in severity and 96% resolved without dose interruption or reduction. The most common AEs were arthralgia (70.5%), injection-site reaction (62.1%), injection-site erythema (47.9%), and headache (47.1%). Acute systemic hypersensitivity events consistent with clinical National Institute of Allergy and Infectious Diseases and the Food Allergy and Anaphylaxis Network anaphylaxis criteria were observed in 12 participants (17 events); of these, 6 participants remained on treatment. Acute systemic hypersensitivity events including potential events of anaphylaxis were not associated with immunoglobulin E, and all events resolved without sequelae.
Results from the PRISM Phase 3 program support the efficacy of pegvaliase for the treatment of adults with PKU, with a manageable safety profile in most participants. The PRISM-2 extension study will continue to assess the long-term effects of pegvaliase treatment.
Phenylalanine ammonia‐lyase (PAL) is a crucial enzyme for various biotechnology applications, such as producing phenols, antioxidants, and nutraceuticals. However, feedback inhibition from its ...product, cinnamic acid, limits its forward reaction rate. Therefore, this study aims to address the feedback inhibition in PAL using enzyme engineering strategies. Random and site‐directed mutagenesis approaches were utilized to screen mutant enzymes with ameliorated tolerance against cinnamic acid. A thermotolerant and cinnamate‐tolerant mutant was rationally identified using a high throughput screening method and subsequent biochemical characterization. We evaluated cinnamate affinity among the seven rationally selected mutations, and the T102E mutation was identified as the most promising mutant. This mutant showed a six‐fold reduction in the affinity of PAL for cinnamic acid and a two‐fold increase in operational stability compared with native PAL. Furthermore, the enzyme was immobilized on carbon nanotubes to increase its robustness and reusability. The immobilized mutant PAL showed greater efficiency in the deamination of phenylalanine present in protein hydrolysate than its free form. The rationale behind the enhancement of cinnamate tolerance was validated using molecular dynamic simulations. Overall, the knowledge of the sequence‐function relationship of PAL was applied to drive enzyme engineering to develop highly tolerant PAL.
Graphical and Lay Summary
PAL is an enzyme used for the conversion of phenylalanine to cinnamate. However, cinnamate accumulation inhibits the forward rate of reaction. A mutant PAL (T102E) was created to overcome this, which showed superior cinnamate tolerance. A biotransformation platform was then developed by immobilising PAL onto carbon nanotubes, enabling its reusability. Further, computer simulations were utilised to comprehend the functioning of the mutant PAL, presenting a potential approach to improve the kinetics of PAL‐based biotransformation.
Ammonia-lyases and aminomutases are mechanistically and structurally diverse enzymes which catalyze the deamination and/or isomerization of amino acids in nature by cleaving or shifting a C–N bond. ...Of the many protein families in which these enzyme activities are found, only a subset have been employed in the synthesis of optically pure fine chemicals or in medical applications. This review covers the natural diversity of these enzymes, highlighting particular enzyme classes that are used within industrial and medical biotechnology. These highlights detail the discovery and mechanistic investigations of these commercially relevant enzymes, along with comparisons of their various applications as stand-alone catalysts, components of artificial biosynthetic pathways and biocatalytic or chemoenzymatic cascades, and therapeutic tools for the potential treatment of various pathologies.
Summary
Sugarcane mosaic virus (SCMV) is a pathogen of worldwide importance that causes dwarf mosaic disease on maize (Zea mays). Until now, few maize genes/proteins have been shown to be involved in ...resistance to SCMV. In this study, we characterized the role of maize phenylalanine ammonia‐lyases (ZmPALs) in accumulation of the defence signal salicylic acid (SA) and in resistance to virus infection. SCMV infection significantly increased SA accumulation and expression of SA‐responsive pathogenesis‐related protein genes (PRs). Interestingly, exogenous SA treatment decreased SCMV accumulation and enhanced resistance. Both reverse transcription‐coupled quantitative PCR and RNA‐Seq data confirmed that expression levels of at least four ZmPAL genes were significantly up‐regulated upon SCMV infection. Knockdown of ZmPAL expression led to enhanced SCMV infection symptom severity and virus multiplication, and simultaneously resulted in decreased SA accumulation and PR gene expression. Intriguingly, application of exogenous SA to SCMV‐infected ZmPAL‐silenced maize plants decreased SCMV accumulation, showing that ZmPALs are required for SA‐mediated resistance to SCMV infection. In addition, lignin measurements and metabolomic analysis showed that ZmPALs are also involved in SCMV‐induced lignin accumulation and synthesis of other secondary metabolites via the phenylpropanoid pathway. In summary, our results indicate that ZmPALs are required for SA accumulation in maize and are involved in resistance to virus infection by limiting virus accumulation and moderating symptom severity.
Pegvaliase is a recombinant Anabaena variabilis phenylalanine ammonia lyase (PAL) enzyme under investigation for treatment of adult phenylketonuria (PKU). This manuscript describes results of a ...randomized discontinuation trial (RDT) designed to evaluate the effects of pegvaliase treatment on blood phenylalanine (Phe) and neuropsychiatric outcomes in adults with PKU.
PRISM-2 is a 4-part, Phase 3 study that enrolled adults with PKU receiving pegvaliase treatment (initiated in a prior Phase 2 or Phase 3 study). The RDT, Part 2 of PRISM-2, was an 8-week trial that evaluated change in blood Phe concentrations, neuropsychiatric and neurocognitive measures, and safety outcomes in PRISM-2 participants who had achieved at least a 20% blood Phe reduction from pre-treatment baseline with pegvaliase treatment. Participants were randomized 2:1 to either continue pegvaliase (20 mg/day or 40 mg/day) or switch to matching placebo.
The pooled pegvaliase group enrolled 66 participants and each placebo group enrolled 14 participants. The primary endpoint of change in blood Phe concentration from RDT entry to RDT Week 8 was met with clinically meaningful and statistically significant differences between the pegvaliase and placebo groups. Mean (SD) blood Phe at the beginning of the RDT when all participants were receiving pegvaliase was 563.9 μM (504.6) in the group assigned to the 20 mg/day placebo group (n = 14), 508.2 μM (363.7) in those assigned to the 40 mg/day placebo group (n = 14), and 503.9 μM (520.3) in those assigned to continue pegvaliase treatment (n = 58). At Week 8 of the RDT, the least squares mean change (95% confidence interval) in blood Phe was 949.8 μM (760.4 to 1139.1) for the 20 mg/day placebo group and 664.8 μM (465.5 to 864.1) for the 40 mg/day placebo group in comparison to 26.5 μM (−68.3 to 121.3) for the pooled (20 mg/day and 40 mg/day) pegvaliase group (P < 0.0001 for pooled pegvaliase group vs each placebo group). Adverse events (AEs) were usually lower in the pooled placebo group when compared to the pooled pegvaliase group. The most common AEs for the pooled pegvaliase and pooled placebo groups were arthralgia (13.6% and 10.3%, respectively), headache (12.1% and 24.1%), anxiety (10.6% and 6.9%), fatigue (10.6% and 10.3%), and upper respiratory tract infection (1.5% and 17.2%).
Mean blood Phe reduction was sustained in the pegvaliase group, while placebo groups had mean blood Phe concentration increase toward pre-treatment baseline levels. Results from this study confirmed the efficacy of pegvaliase in maintaining reduced blood Phe concentrations with a manageable safety profile for most participants.
An efficient preparative-scale synthetic procedure of l-phenylalanine derivatives has been developed using mutant variants of phenylalanine ammonia-lyase from Arabidopsis thaliana (AtPAL). After ...rigorous reaction engineering, the AtPAL-catalyzed hydroamination reaction of cinnamic acids provided several unnatural amino acids of high synthetic value, such as (S)-m- and (S)-p-methoxyphenylalanine; (S)-o- and (S)-m-methylphenylalanine; and (S)-o- and (S)-p-bromophenylalanine at preparative scale, significantly surpassing the catalytic efficiency in terms of conversions and yields of the previously reported PcPAL-based biotransformations. The AtPAL variants tolerated high substrate and product concentrations, representing an important extension of the PAL-toolbox, while the engineered biocatalytic procedures of improved E-factor and space-time yields fulfill the requirements of sustainable and green chemistry, providing facile access to valuable amino acid building blocks.
Phenylalanine ammonia-lyase (PAL) has a crucial role in secondary phenylpropanoid metabolism and is one of the most extensively studied enzymes with respect to plant responses to biotic and abiotic ...stress. Here, we identified the pepper (Capsicum annuum) PAL (CaPAL1) gene, which was induced in pepper leaves by avirulent Xanthomonas campestris pv. vesicatoria (Xcv) infection. CaPAL1-silenced pepper plants exhibited increased susceptibility to virulent and avirulent Xcv infection. Reactive oxygen species (ROS), hypersensitive cell death, expression of the salicylic acid (SA)-dependent marker gene CaPR1, SA accumulation, and induction of PAL activity were significantly compromised in the CaPAL1-silenced pepper plants during Xcv infection. Overexpression (OX) of CaPAL1 in Arabidopsis conferred increased resistance to Pseudomonas syringae pv. tomato (Pst) and Hyaloperonospora arabidopsidis infection. CaPAL1-OX leaves exhibited restricted Pst growth, increased ROS burst and cell death, and induction of PR1 expression and SA accumulation. The increase in PAL activity in healthy and Pst-infected leaves was higher in CaPAL1-OX plants than in wild-type Arabidopsis. Taken together, these results suggest that CaPAL1 acts as a positive regulator of SA-dependent defence signalling to combat microbial pathogens via its enzymatic activity in the phenylpropanoid pathway.
Phenylalanine ammonia-lyase (PAL) is the first enzyme of the general phenylpropanoid pathway catalyzing the nonoxidative elimination of ammonia from L-phenylalanine to give trans-cinnamate. In ...monocots, PAL also displays tyrosine ammonia lyase (TAL) activity, leading to the formation of p-coumaric acid. The catalytic mechanism and substrate specificity of a major PAL from sorghum (Sorghum bicolor; SbPAL1), a strategic plant for bioenergy production, were deduced from crystal structures, molecular docking, site-directed mutagenesis, and kinetic and thermodynamic analyses. This first crystal structure of a monocotyledonous PAL displayed a unique conformation in its flexible inner loop of the 4-methylidene-imidazole-5-one (MIO) domain compared with that of dicotyledonous plants. The side chain of histidine-123 in the MIO domain dictated the distance between the catalytic MIO prosthetic group created from ¹⁸⁹Ala-Ser-Gly¹⁹¹ residues and the bound L-phenylalanine and L-tyrosine, conferring the deamination reaction through either the Friedel-Crafts or E₂ reaction mechanism. Several recombinant mutant SbPAL1 enzymes were generated via structure-guided mutagenesis, one of which, H123F-SbPAL1, has 6.2 times greater PAL activity without significant TAL activity. Additional PAL isozymes of sorghum were characterized and categorized into three groups. Taken together, this approach identified critical residues and explained substrate preferences among PAL isozymes in sorghum and other monocots, which can serve as the basis for the engineering of plants with enhanced biomass conversion properties, disease resistance, or nutritional quality.
Salicylic acid (SA), an essential regulator of plant defense, is derived from chorismate via either the phenylalanine ammonia lyase (PAL) or the isochorismate synthase (ICS) catalyzed steps. The ICS ...pathway is thought to be the primary contributor of defense-related SA, at least in Arabidopsis. We investigated the relative contributions of PAL and ICS to defenserelated SA accumulation in soybean (Glycine max).
Soybean plants silenced for five PAL isoforms or two ICS isoforms were analyzed for SA concentrations and SA-derived defense responses to the hemibiotrophic pathogens Pseudomonas syringae and Phytophthora sojae.
We show that, unlike in Arabidopsis, PAL and ICS pathways are equally important for pathogen-induced SA biosynthesis in soybean. Knock-down of either pathway shuts down SA biosynthesis and abrogates pathogen resistance. Moreover, unlike in Arabidopsis, pathogen infection is associated with the suppression of ICS gene expression. Pathogen-induced biosynthesis of SA via the PAL pathway correlates inversely with phenylalanine concentrations. Although infections with either virulent or avirulent strains of the pathogens increase SA concentrations, resistance protein-mediated response to avirulent P. sojae strains may function in an SA-independent manner.
These results show that PAL- and ICS-catalyzed reactions function cooperatively in soybean defense and highlight the importance of PAL in pathogen-induced SA biosynthesis.
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